ES2657930T3 - New derivatives of peptides as antibiotics - Google Patents

Abstract

Compound of formula (I): Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-NH- (CH2) nR (I) in which Xaa1, Xaa2, Xaa3, Xaa8 and Xaa10 are selected independently of the group consisting of lysine, 3-hydroxylysine, 4-hydroxylysine, 5-hydroxylysine, 3,4-dihydroxylysine, 3,5-dihydroxylysine, 4,5-dihydroxylysine, ornithine, 3-hydroxyiornithine, 4-hydroxyiornithine, 3,4-dihydroxyiornithine, 2,4-diaminobutanoic acid, 3-hydroxy-2,4-diaminobutanoic acid, arginine, histidine, serine, and threonine; Xaa4 is glycine; Xaa5 is ornithine Xaa6 is proline, 3-hydroxyproline, 4-hydroxyproline, aziridin-2-carboxylic acid, azetidin-2-carboxylic acid, pipecolic acid, 4-oxaproline, 3-thiaproline, 4-thiaproline, 3,4-dehydroproline, 4-aminoproline, 4-fluoroproline, α-methylproline, or α-allylproline; Xaa7 is histidine Xaa9 is arginine, 2,3-dehydroarginine, citrulline, 2,3-dehydrocitrulline, canavanine, or 2,3-dehydrocanavanine; n is 2, 3, 4, 5, 6, 7, 8, 9, or 10; and R is -OH, -NH2 or -COOH.

Description

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In some embodiments, the odilomycins are isolated.

In some embodiments, the odilomycins have a purity greater than about 50%. In some embodiments, the odilomycins have a purity greater than about 60%. In some embodiments, the odilomycins have a purity greater than about 70%. In some embodiments, the odilomycins have a purity greater than about 80%. In some embodiments, the odilomycins have a purity greater than about 85%. In some embodiments, the odilomycins have a purity greater than about 90%. In some embodiments, the odilomycins have a purity greater than about 95%. In some embodiments, the odilomycins have a purity greater than about 98%. In some embodiments, the odilomycins have a purity greater than about 99%. In some embodiments, any established purity values may constitute a lower and / or higher extreme value of a purity range where appropriate or in any of the lower limits may be combined with any of the upper limits.

In some embodiments, the odilomycins and / or compositions comprising them are useful as a medicament, as an antibiotic, as an antimicrobial or in the treatment of microbial diseases, in particular of a bacterial infection caused, for example, by pathogenic bacteria.

In some embodiments, the odilomycins and / or compositions comprising them are useful in the treatment of bacterial infections, for example in the treatment of an in-hospital infection or a nosocomial bacterial infection.

In some embodiments, the present invention provides a method of treating a subject suffering from an in-hospital bacterial infection comprising administering to said subject an effective amount of a compound of formula (I).

In some embodiments, the present invention provides a method of treating a subject suffering from a nosocomial bacterial infection comprising administering to said subject an effective amount of a compound of formula (I).

In one aspect, the invention also relates to the use of compounds of formula (I) for the manufacture of a medicament for treating a microbial infection or a microbial disease.

In another aspect, the invention also relates to the use of compounds of formula (I) for the manufacture of an antibiotic composition.

In yet another aspect, the present invention also relates to treatment methods comprising the administration of a compound of formula (I), or pharmaceutical compositions comprising a compound of formula (I), to a subject in need thereof. Odilomycins and / or compositions comprising them may be useful, for example, in the treatment, suppression and / or prevention of an infection and / or bacterial disease.

In some embodiments, the invention provides a method of treatment, prevention and / or suppression of a bacterial infection comprising administration to a subject in need thereof of a therapeutically effective amount of a compound of formula (I) or a composition which comprises a compound of formula (I).

In some embodiments, the present invention provides a method of treating a subject suffering from a bacterial infection comprising administering to said subject a compound of formula (I).

In some embodiments, the present invention provides a method for suppressing a bacterial infection in a subject comprising administering to the subject a compound of formula (I).

In some further embodiments, the present invention provides a method of treating a subject suffering from a multi-drug resistant bacterial infection comprising administering to said subject an effective amount of a compound of formula (I).

In some embodiments, the methods of the present invention provide for the inhibition of bacteria that are resistant to other drugs or antibiotics, or related infection. In some embodiments, the procedures provide treatment, suppression and / or prevention of infection by multi-drug resistant bacteria.

In some embodiments, the bacterial infection is resistant to multiple drugs. In some embodiments, the bacterial strain is in-hospital. In some embodiments, the bacterial strain is

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nosocomial

In some embodiments, the bacterial infection comprises infection by Gram-negative bacteria. In some embodiments, the bacterial infection comprises infection by Gram-positive bacteria. In some embodiments, the bacterial infection comprises infection by more than one bacterial strain.

In some embodiments, the bacterial or microbial infection is an infection caused in whole or in part by bacteria of the Achromobacter, Actinobacillus, Actinomyces, Acinetobacter, Aeromonas, Anaplasma, Bacillus, Bacteroides, Bartonella, Bdellovibrio, Bifidobacterium, Bordetella families, Borrelia, Brucella, Burkholderia, Campylobacter, Capnocytophaga, Cardiobacterium, Chlamydia, Chlamydophila, Chromobacterium, Citrobacter, Clostridium, Corynebacterium, Coxiella, Ehrlichia, Enterobacter, Enterococcus, Erysipelothrix, Escherichia, Francisella, Fusobacterium, Haemophilus, Helicobacter, Hemobartonella, Klebsiella, Lactobacillus, Legionella, Leptospira, Listeria, Mannheimia, Moraxella, Morganella, Mycobacterium, Mycoplasma, Neisseria, Neorickettsia, Nocardia, Pasteurella, Peptostreptococcus, Photorhabdus, Porphyromonas, Prevotella, Propionibacterium, Proteus, Pseudraumia, Pseudraumia, Pseudomia, Serse Spill, Pseudomia, Serse Spillia, Pseudomia, Serse Staphylococcus, Stenot rophomonas, Streptobacillus, Streptococcus, Treponema, Tropheryma, Ureaplasma, Vibrio or Yersinia.

In some embodiments, the bacterial or microbial infection is an infection caused in whole or in part by Acinetobacter baumannii, Bacillus subtilis, Burkholderia cepacia, Enterobacter clocae, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Staphylococcusotomonascumus, Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus. maltophilia, Serratia marescens or Pseudomonas aeruginosa. Preferably, the strain of Xenorhabdus nematophila CNCM 1-4530, the culture supernatant of this strain and the cell extracts of this strain show antibiotic activity against Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Klebsiella pneumonia, Klebsiella oxytocacterium Acinetocacterium, Acinetobacter bacteria , Escherichia coli, Moraxella catarrhalis, Pseudomonas aeruginos and Stenotrophomonas maltophilia.

In some embodiments, the disclosure provides the use of pharmaceutical compositions and / or medicaments comprised of the compounds of formula (I) in a method of treating a bacterial infection and / or pathological condition and / or disorder caused by said bacterial infection. or related to it.

In some embodiments, the methods comprise administering to the subject an effective amount of a compound of formula (I) alone or in combination with a second antibiotic compound, or a composition comprising a compound of formula (I) alone or in combination with a second antibiotic compound and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known to those skilled in the art and include, for example, adjuvants, diluents, excipients, fillers and lubricants. Often, the pharmaceutically acceptable carrier is chemically inert against active compounds and is not toxic under the conditions of use. Examples of pharmaceutically acceptable carriers may include, for example, water or saline solution, polymers such as polyethylene glycol, carbohydrates and derivatives thereof, oils, fatty acids or alcohols.

In some embodiments, the method of treatment, prevention and / or suppression of a disorder related to a bacterial infection comprises the steps of: (i) identifying a subject in need of said treatment; (ii) providing a compound of formula (I) alone or in combination with a second antibiotic compound, or a composition comprising a compound of formula (I) alone or in combination with a second antibiotic compound and a pharmaceutically acceptable carrier and (iii ) administering said compound or said compounds or said composition in a therapeutically acceptable amount to treat, prevent and / or suppress a bacterial infection in a subject in need of said treatment.

In some embodiments, the methods comprise administering to the subject an effective amount of a compound of formula (I); or a composition comprising a compound of formula (I) and a pharmaceutically acceptable carrier.

In some embodiments, the method of treatment, prevention and / or suppression of a disorder related to a bacterial infection comprises the steps of: (i) identifying a subject in need of said treatment; (ii) providing a compound of formula (I) or a composition comprising a compound of formula (I) and a pharmaceutically acceptable carrier and (iii) administering said compound or said composition in a therapeutically effective amount to treat, prevent and / or suppress the pathological state or disorder related to a bacterial infection in a subject in need of such treatment.

In some embodiments, treatment refers, in general, to treatment and therapy, both of a human being and of an animal (for example, in veterinary applications), with which some desired therapeutic effect is achieved, for example, inhibition of the progress of the disorder. Treatment may also include, but not limited to, a reduction in the speed of progression, a stop in the speed of progression.

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progression, an improvement of the disorder, the cure of the disorder, a stabilized (i.e. not worsened) state of the disease or condition, prevent the spread of the disease or condition, the delay or slowdown of the progression of the disease or condition , an improvement or palliation of the pathological state or condition and remission (both partial and total), both detectable and undetectable. In some embodiments, treatment may also mean prolonging survival compared to expected survival if no treatment is received. In some embodiments, a treatment is also included as a prophylactic measure (i.e. prevention). For example, the use with subjects who have not yet developed the disorder, but who are at risk of developing the disorder, is understood as the term "treatment."

In some embodiments, the treatment comprises combination treatments and polytherapies, in which two or more treatments or therapies are combined, for example sequentially or simultaneously. For example, the active agents of the present invention can also be used in other polytherapies, for example in conjunction with other agents, for example, other antimicrobials or other antibiotics, etc.

The subjects are found in in vitro and in vivo systems, including, for example, isolated or cultured cells or tissues, non-cellular and animal in vitro test systems (eg, an amphibian, a bird, a fish, a mammal, a marsupial, a human being, a pet such as, for example, a cat, a dog, a monkey, a mouse or a rat; or a useful animal such as, for example, a horse, a bovine (such as a cow ), a turkey, a chicken or a pig).

In some embodiments, the subject is a mammal. In some embodiments, the subject is a bird, a pig, a bovine or a human being. In some embodiments, the subject is a human being. In some embodiments, the subject is a bird. In some embodiments, the subject is a pig or a pig. In some embodiments, the subject is a turkey or a chicken.

In addition, the compositions or processes may additionally comprise one or more additional antibacterial or antibiotic compounds in combination with a compound of formula (I) alone. Examples of such compounds include, but are not limited to, daptomycin, oxacillin, piperacillin / tazobactam, ticaricillin / clavulanic acid, amoxicillin / clavulanic acid, erythromycin, cefepim, clindamycin, imipenem, gentamicin, ciprofloxacin, aztreonamine, vancomycin, kincomancin, vancomycin, kamycinamine, vancomycinamine , ampicillin, tetracycline and the like.

In some embodiments, the additional antibiotic (or second antibiotic) is an aminoglycoside antibiotic. Aminoglycoside antibiotics are antibiotic compounds in which a portion of a molecule contains an amino-modified sugar. Examples of aminoglycoside antibiotics include but are not Disposal The amikacin, apramycin, arbekacin, astromicina, bekanamycin, capreomycin, dibekacin, dihydrostreptomycin, elsamitrucin, G418, gentamycin, hygromycin B, isepamicin, kanamycin, kasugamycin, micronomicina, neomycin, netilmicin sulfate paromomycin, ribostamycin, sisomycin, streptoduocin, streptomycin, tobramycin and verdamicin.

Thus, in some embodiments the methods and / or compositions further comprise one or more additional antibacterial compounds in combination with a compound of formula (I). In some embodiments, the additional antibacterial compound is selected from the group consisting of daptomycin, oxacillin, piperacillin / tazobactam, ticaricillin / clavulanic acid, amoxicillin / clavulanic acid, erythromycin, cefepim, clindamycin, imipenem, gentamicin, ciprofonamine, aziprofonamine , linezolid, rifampicin, kanamycin, ampicillin and tetracycline. In some embodiments, the additional antibacterial is kanamycin. In some embodiments, the additional antibiotic is an aminoglycoside antibiotic.

In some embodiments, the present invention provides a method for treating, suppressing and / or preventing a bacterial infection by the combined use of the compound of formula (I) and a second antibiotic compound in subjects in which the use of the compound of formula (I) or the second antibiotic compound alone does not provide the desired therapeutic effect. Surprisingly, it has been found that when other antibiotic compounds are used together with a compound of formula (I), statistically significant increases in antibiotic effects are observed. In some embodiments, there is a synergistic effect between a second antibiotic compound, such as kanamycin, and the compound of formula (I). Thus, in some embodiments, the kanamycin and the compound of formula (I) are administered in amounts that show a synergistic reduction of bacterial levels. In some embodiments, the kanamycin and the compound of formula (I) are administered in amounts that show synergistic treatment, synergistic suppression and / or synergistic prevention of a bacterial infection.

In another aspect, the present invention is a pharmaceutical formulation comprising an effective amount of a compound of formula (I). In some embodiments, Xaa1 is lysine.

In some embodiments, Xaa2 is 3-hydroxy-2,4-diaminobutanoic acid.

In some embodiments, Xaa3 is 3-hydroxy-2,4-diaminobutanoic acid.

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memory.

It will also be recognized that any or all combinations, embodiments and aspects of the invention may be combined in any way to provide other combinations, embodiments and aspects within the scope of the invention unless otherwise it is not possible .

The invention will be described in more detail by the following non-limiting examples.

Examples

Example 1: Production and fermentation

Producer body

The Xenorhabdus nematophila CNCM 1-4530 ("Diversité, genomes et interactions microorganismsinsectes" collection) was grown in the middle of Luria-Bertani (LB, consisting of bactotriptone, 10 g / l, yeast extract, 5 g / l, and NaCl, 10 g / l) for a liquid culture and in LB-agar for solid cultures. The phase (I or II) status of this strain was determined by culturing in NBTA (nutrient agar (Difco), 31 g / l, bromothymol blue, 25 mg / l, and 2,3,5-triphenyltetrazolium chloride at 1 %, 40 mg / l) and measuring antibacterial activity against Micrococcus luteus. Xenorhabdus shows two forms or variants of colonies when grown in vitro. Modifications of the outer membrane induce differential adsorption of dyes by variants. Phase I variants absorb dyes and are blue in NBTA plates, while phase II colonies are red. Phases I and II of the strains are indicated with suffixes (/ 1 and / 2, respectively) attached to the denominations of the strains. This strain was maintained at 15 ° C in NBTA medium.

Fermentation

The Xenorhabdus nematophila CNCM 1-4530 was grown for 72 h, at 28 ° C with shaking in a 2 l Erlenmeyer flask containing 500 ml of culture medium composed of bactopeptone, 15 g / l, MgSO4 · 7H2O, 2 g / l, and glucose, 2 g / l. 0.1% (v / v) of a 24-hour preculture was inoculated in the same medium. Antibiotic production was monitored by analytical HPLC.

Example 2: Isolation

Bacterial cells were removed by low speed centrifugation (6000 × g, 10 min at 4 ° C) and the supernatant was sterilized on a 0.22 µm pore size filter. The supernatant was added (1: 1; v / v) to a 0.02 M 0.1 M-Tris NaCl buffer (pH 7) and subjected to ion exchange chromatography in a CarboxyMethyl Sep Pack cartridge (Accell Plus CM, Waters). Unbound material was removed by washing with 0.1 M NaCl-0.02 M buffer (pH 7) and antibiotic activity products were eluted with 0.1 M Na-Tris buffer 0.02 M (pH 7). This eluate was acidified with 0.1% (v / v) trifluoroacetic acid (TFA) and then subjected to reverse phase chromatography on a Sep Pack C18 cartridge (Sep-Pak Plus C18, Waters). Unbound material was removed by washing with 0.1% H2O-TFA and the combined antibiotics were eluted with acetonitrile. The eluate was lyophilized and then resuspended in water (1: 5; v / v). The pure compounds were isolated from the crude extract by reverse phase HPLC using a C18 column (Waters; Symmetry Symmetry C18; 5 µm; 4.6X150 mm), a linear gradient of H2O / 0.1% TFA-acetonitrile starting at 0 % at 30% in 30 min, a flow rate of 1 ml / min and a UV detection of 200 to 400 nm, providing odilomycins with the following HPLC retention times: Odilomycin A: 14.16 min (purity: 98% UV) , Odilomycin B: 14.44 min (purity: 95% UV) and Odilomycin C: 14.6 min (purity: 94% UV).

Example 3: Characterization and physicochemical properties

NMR and MS analysis

The purified compound was analyzed by mass spectroscopy and NMR to determine its chemical structure.

The NMR study was carried out on a Bruker Advance spectrometer that operated at 700 MHz equipped with a cryoprobe. The sample (10 mM) was dissolved in water (95/5 H2O / D2O v / v) and the pH adjusted to 3.5 with hydrochloric acid. All data was recorded at 280 ° K. Proton chemical shifts were expressed with respect to sodium 4,4-dimethyl-silapentane-1-sulphonate, according to the recommendations of the IUPAC. The double quantum filter correlation spectroscopy spectra (DQF-COZY), z-filter total correlation spectroscopy (z-TOCSY) and nuclear Overhauser effect spectroscopy (NOESY) were recorded in a phase sensitive mode, using the States-TPPI procedure (Marion D. Et al, J. Magn. Reson. 85, 393-399 (1989)). The z-TOCSY spectra were obtained with a mixing time of 80 ms and the NOESY spectra with mixing times of 220 ms. The HSQC experiments of 1H-13C and HSQC-TOCSY of 1H-13C were carried out with the same sample. The water resonance established in the vehicle frequency is

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Spin system

Group 1H (ppm) 13C (ppm)

CβH2 CγH2 CδH2 CεH2

1.66 / 1.62 1.2 1.45 2.78 30.4 21.1 26.3 39.0

Hydroxy-diamino-butyric acid2 Dab (βOH) 2

HN CαH CpH-OH CγH2 NH2 8.83 4.28 3.89 2.99 / 2.79 -56.3 67.4 41.9

Hydroxy-diamino-butyric acid3 Dab (βOH) 3

HN CαH CβH-OH CγH2 NH2 8.63 4.33 3.93 2.98 / 2.80 - -56.3 67.7 41.5 -

Gly4

HN CαH 8.37 3.8 -42.1

Ornithine5 Orn5

HN CαH CβH2 CγH2 CδH2 NH2 8.15 4.46 1.58 / 1.42 1.49 / 1.4 2.74 - -51.2 27.5 23.2 39 -

Pro6

CαH CβH2 CγH2 CδH2 4.10 1.98 / 1.60 1.72 3.48 / 3.38 60.4 29.5 24.3 47.7

His7

HN CαH CβH2 Cδ2H Cε1H 8.49 4.46 2.98-2.91 7.03 8.33 -52.0 26.3 118 134

δ Hydroxylysine8 Dhl8

HN CαH CβH2 CγH2 CδH-OH CεH2 NH2 8.41 4.12 1.70 1.36 / 1.26 3.62 2.86 / 2.63 - -53.8 27.0 30.0 67.0 44.5 -

dehydroarginine9 Dha9

HN Cα CβHCγH2 CδH2 HNε C (NH2) = NH 9.6 -6.17 2.20 3.10 7.0 - --132 26.5 39.5 -

δ-Hydroxylysine10 Dhl10

HN CαH CβH2 CγH2 CδH-OH CεH2 NH2 8.05 4.06 1.65 1.27 3.60 2.86 / 2.63 - 54.0 27.0 30.0 67.0 44.5 -

Diaminobutane11 Dbt11

HN CαH2 8.05 2.95 39.5

CβH2

1.30 25.3

CγH2

1.40 24.0

CδH2

2.78 39.0

NH2

- -

Table 2. Chemical displacements of odilomycin B (water, 280 K)

Spin system

Group 1H (ppm) 13C (ppm)

Lys1

HN CαH CβH2 CγH2 CδH2 CεH2 -3.8 1.66 / 1.62 1.2 1.45 2.78 -53.0 30.4 21.1 26.3 39.0

Hydroxy-diamino-butyric acid2 Dab (βOH) 2

HN CαH CβH-OH CγH2 NH2 8.83 4.28 3.89 2.99 / 2.79 -56.3 67.4 41.9

Hydroxy-diamino-butyric acid3 Dab (βOH) 3

HN CαH CβH-OH CγH2 NH2 8.63 4.33 3.93 2.98 / 2.80 - -56.3 67.7 41.5 -

Gly4

HN CαH 8.37 3.8 -42.1

Ornithine5 Orn5

HN CαH CβH2 CγH2 CδH2 NH2 8.15 4.46 1.58 / 1.42 1.49 / 1.4 2.74 - -51.2 27.5 23.2 39 -

Pro6

CαH CβH2 CγH2 CδH2 4.10 1.98 / 1.60 1.72 3.48 / 3.38 60.4 29.5 24.3 47.7

HN

8.49 -

CαH

4.46 52.0

His7

CβH2 2.98-2.91 26.3

Cδ2H

7.03 118

Cε1H

8.33 134

Lys8

HN CαH CβH2 CγH2 Cδ H2 CεH2 NH2 8.41 4.12 1.70 1.36 / 1.26 1.53 2.86 / 2.63 - -53.8 27.0 30.0 26.4 44.5 -

dehydroarginine9 Dha9

HN Cα CβHCγH2 CδH2 HNε C (NH2) = NH 9.6 -6.17 2.20 3.10 7.0 - --132 26.5 39.5 -

δ-Hydroxylysine10

HN 8.05

Spin system

Group 1H (ppm) 13C (ppm)

DHL10

CαH 4.06 54.0

CβH2 CγH2

1.65 1.27 27.0 30.0

CδH-OH

3.60 67.0

CεH2 NH2

2.86 / 2.63 - 44.5 -

HN

8.05

CαH2

2.95 39.5

Diaminobutane11 Dbt11

CβH2 CγH2 1.30 1.40 25.3 24.0

CδH2

2.78 39.0

NH2

- -

Table 3. Chemical displacements of odilomycin C (water, 280 K)

Spin system

Group 1H (ppm) 13C (ppm)

Lys1

HN CαH CβH2 CγH2 CδH2 CεH2 -3.8 1.66 / 1.62 1.2 1.45 2.78 -53.0 30.4 21.1 26.3 39.0

HN

8.83 -

Hydroxy-diamino-butyric acid2 Dab (βOH) 2

CαH CβH-OH 4.28 3.89 56.3 67.4

CγH2

2.99 / 2.79 41.9

NH2

Hydroxy-diamino-butyric acid3 Dab (βOH) 3

HN CαH CβH-OH CγH2 NH2 8.63 4.33 3.93 2.98 / 2.80 - -56.3 67.7 41.5 -

Gly4

HN CαH 8.37 3.8 -42.1

Ornithine5 Orn5

HN CαH CβH2 CγH2 CδH2 NH2 8.15 4.46 1.58 / 1.42 1.49 / 1.4 2.74 - -51.2 27.5 23.2 39 -

Pro6

CαH CβH2 CγH2 CδH2 4.10 1.98 / 1.60 1.72 3.48 / 3.38 60.4 29.5 24.3 47.7

HN

8.49 -

CαH

4.46 52.0

His7

CβH2 2.98-2.91 26.3

Cδ2H

7.03 118

Cε1H

8.33 134

HN

8.41 -

CαH

4.12 53.8

Lys8

CβH2 CγH2 1.70 1.36 / 1.26 27.0 30.0

CδH2

1.53 26.4

CεH2

2.86 / 2.63 44.5

Spin system

Group 1H (ppm) 13C (ppm)

NH2

- -

dehydroarginine 9 Dha9

HN Cα CβHCγH2 CδH2 HNε C (NH2) = NH 9.6 -6.17 2.20 3.10 7.0 --132 26.5 39.5 -

Lys10

HN CαH CβH2 CγH2 CδH-OH CεH2 NH2 8.05 4.06 1.65 1.27 1.58 2.86 / 2.63 - 54.0 27.0 30.0 26.3 44.5 -

Diaminobutane11 Dbt11

HN CαH2 CβH2 CγH2 CδH2 NH2 8.05 2.95 1.30 1.40 2.78 - 39.5 25.3 24.0 39.0 -

Table 4. Key fragments of the 1297 [M + H] + odilomycin A ions

172

[Gly4 / Orn5] +

233

[Dab (βOH) 2 / Dab (βOH) 3] +

235

[Pro6 / His7] +

245

[Lys1 / Dab (βOH) 2] +

282

[His7 / Dhl8] +

299

[DHL8 / Dha9] + or [Dha9 / DHL10] +

361

[Lys1 / Dab (βOH) 2 / Dab (βOH) 3] +

370

[Dha9 / Dhl10 / Dbt11] + -NH3 +

379

[Pro6 / His7 / Dhl8] +

396

[Pro6 / His7 / Dhl8] + + NH3 +

406

[Gly4 / Orn5 // Pro6 / His7] +

436

[HiS7 / Dhl8 / Dha9] +

530

[Dhl8 / Dha9 / Dhl10 / Dbt11] +

533

[Lys1 / Dab (βOH) 2 / Dab (βOH) 3 / Gly4 / Orn5] + or [Pro6 / His7 / Dhl8 / Dha9] +

550

[Gly4 / Orn5 / Pro6 / His7 / Dhl8] +

567

[Gly4 / Orn5 / Pro6 / His7 / Dhl8] + + NH3 +

683

[Dab (βOH) 3 / Gly4 / Orn5 / Pro6 / His7 / Dhl8] + + NH3 +

704

[Gly4 / Orn5 / Pro6 / His7 / Dhl8 / Dha9] +

765

[Pro6 / His7 / Dhl8 / Dha9 / Dhl10 / Dbt11] +

820

[Dab (βOH) 3 / Gly4 / Orn5 / Pro6 / His7 / Dhl8 / Dha9] +

936

[Gly4 / Orn5 / Pro6 / His7 / Dhl9 / Dha9 / Dhl10 / Dbt11] +

964

[Dab (βOH) 3 / Gly4 / Orn5 / Pro6 / His7 / Dhl8 / Dha9 / Dhl10] +

1052

[Dab (βOH) 3 / Gly4 / Orn5 / Pro6 / His7 / Dhl8 / Dha9 / Dhl10 / Dbt11] +

1064

[Lys1 / Dab (βOH) 2 / Dab (βOH) 3 / Gly4 / Orn5 / Pro6 / His7 / Dhl8 / Dha9] +

1297

[Lys1 / Dab (βOH) 2 / Dab (βOH) 3 / Gly4 / Orn5 / Pro6 / His7 / Dhl8 / Dha9 / Dhl10 / Dbt11] +

Table 5. Key fragments of the 1281 [M + H] + odilomycin B ions

172

[Gly4 / Orn5] +

233

[Dab (βOH) 2 / Dab (βOH) 3] +

235

[Pro6 / His7] +

245

[Lys1 / Dab (βOH) 2] +

266

[His7 / Lys8] +

361

[Lys1 / Dab (βOH) 2 / Dab (βOH) 3] +

370

[Dha9 / Dhl10 / Dbt11] + -NH3 +

363

[Pro6 / His7 / Lys8] +

380

[Pro6 / His7 / Lys8] + + NH3 +

406

[Gly4 / Orn5 / Pro6 / His7] +

514

[Lys8 / Dha9 / Dhl10 / Dbt11] +

517

[Pro6 / His7 / Lys8 / Dha9] +

533

[Lys1 / Dab (βOH) 2 / Dab (βOH) 3 / Gly4 / Orn5] +

534

[Gly4 / Orn5 / Pro6 / His7 / Lys] +

551

[Gly4 / Orn5 / Pro6 / His7 / Lys8] + + NH3 +

667

[Dab (βOH) 3 / Gly4 / Orn5 / Pro6 / His7 / Lys8] + + NH3 +

688

[Gly4 / Orn5 / Pro6 / His7 / Lys8 / Dha9] +

749

[Pro6 / His7 / Lys8 / Dha9 / Dhl10 / Dbt11] +

804

[Dab (βOH) 3 / Gly4 / Orn5 / Pro6 / His7 / Lys8 / Dha9] +

920

[Gly4 / Orn5 / Pro6 / His7 / Lys8 / Dha9 / Dhl10 / Dbt11] +

948

[Dab (βOH) 3 / Gly4 / Orn5 / Pro6 / His7 / Lys8 / Dha9 / Dhl10] +

1036

[Dab (β0H) 3 / Gly4 / Orn5 / Pro6 / His7 / Lys8 / Dha9 / Dhl10 / Dbt11] +

1048

[Lys1 / Dab (βOH) 2 / Dab (βOH) 3 / Gly4 / Orn5 / Pro6 / His7 / lys8 / Dha9] +

1281

[Lys1 / Dab (βOH) 2 / Dab (βOH) 3 / Gly4 / Orn5 / Pro6 / His7 / lys8 / Dha9 / Dhl10 / Dbt11] +

Table 6. Key fragments of 1264 [M + H] + odilomycin C ions

172

[Gly4 / Orn5] +

233

[Dab (βOH) 2 / Dab (βOH) 3] +

235

[Pro6 / His7] +

245

[Lys1 / Dab (βOH) 2] +

266

[His7 / Lys8] +

361

[Lys1 / Dab (βOH) 2 / Dab (βOH) 3] +

354

[Dha9 / Lys10 / Dbt11] + -NH3 +

363

[Pro6 / His7 / Lys8] +

380

[Pro6 / His7 / Lys8] + + NH3 +

406

[Gly4 / Orn5 / Pro6 / His7] +

498

[Lys8 / Dha9 / Lys10 / Dbt11] +

517

[Pro6 / His7 / Lys8 / Dha9] +

533

[Lys1 / Dab (βOH) 2 / Dab (βOH) 3 / Gly4 / Orn5] +

534

[Gly4 / Orn5 / Pro6 / His7 / Lys8] +

551

[Gly4 / Orn5 / Pro6 / His7 / Lys8] + + NH3 +

688

[Gly4 / Orn5 / Pro6 / His7 / Lys8 / Dha9] +

733

[Pro6 / His7 / Lys8 / Dha9 / Lys10 / Dbt11] +

804

[Dab (βOH) 3 / Gly4 / Orn5 / Pro6 / His7 / Lys8 / Dha9] +

904

[Gly4 / Orn5 / Pro6 / His7 / Lys9 / Dha9 / Lys10 / Dbt11] +

932

[Dab (βOH) 3 / Gly4 / Orn5 / Pro6 / His7 / Lys8 / Dha9 / Lys10] +

1020

[Dab (βOH) 3 / Gly4 / Orn5 / Pro6 / His7 / Lys8 / Dha9 / Lys10 / Dbt11] +

1048

[Lys1 / Dab (βOH) 2 / Dab (βOH) 3 / Gly4 / Orn5 / Pro6 / His7 / lys8 / Dha9] +

1265

[Lys1 / Dab (βOH) 2 / Dab (βOH) 3 / Gly4 / Orn5 / Pro6 / His7 / lys8 / Dha9 / Lys10 / Dbt11] +

5 Example 4: In vitro studies Antibacterial susceptibility test procedures Minimum inhibitory concentrations (MIC) were determined according to the Clinical conditions guidelines

10 and Laboratory Standards Institute (CLSI) detailed in Table 7. The tests were performed in triplicate.

Table 7. MIC determination parameters

Strain (s)

Relevant CLSI Guidelines Culture media and incubation conditions

Acinetobacter baumannii Bacillus subtilis Enterobacter cloacae Enterococcus faecalis Enterococcus faecium Escherichia coli Klebsiella pneumonia Klebsiella oxytoca Pseudomonas aeruginosa Staphylococcus aureus Staphylococcus epidermidis Stenotphomphonia

M07-A8. Antimicrobial dilution procedures Susceptibility tests for bacteria that grow aerobically; approved standard - eighth edition Mueller-Hinton Broth (MH) 37ºC aerobic 18-24 h

Streptococcus pneumonia Streptococcus pyogenes

M07-A8 MH supplemented with 5% lysed horse blood (MHB) 37ºC aerobic 18-24 h

Haemophilus influenzae

M07-A8 Hemophilic test medium (HTM) 37ºC aerobic 18-24 h.

Moraxella catarrhalis

M45-A2 Procedures for antimicrobial dilution and disc susceptibility testing of non-frequently isolated or demanding bacteria; approved guideline - second edition MH 37ºC aerobic 24 h.

Pasteurella Multocida

M45-A2 MHB 37ºC aerobic 24 h.

5

10

fifteen

twenty

25

30

35

40

Mannheimia haemolytica

Not available, followed by M07-A8 MHB 37ºC aerobic 24 h.

Proprionibacterium acnes Bacteroides fragilis

M11-A7. Procedures for the test of antimicrobial susceptibility of anaerobic bacteria; approved standard - seventh edition Schaedler broth supplemented with 1 µg / ml of vitamin K1 and 5% of lysed horse blood (SB) 37ºC aerobic 48 h.

Preparation of inoculums. Five to ten isolated colonies were taken in wells and resuspended in 3 ml of sterile saline. The inoculum was resuspended by vigorous stirring in a vortex mixer for 15 s. Turbidity was adjusted to a 0.5 McFarland standard (1-5 x 106 CFU / ml). The inoculum was subsequently diluted in the appropriate medium (table 7) giving a final inoculum in each well of -2-8 x 105 CFU / ml.

Addition of the test article. Stock solutions of odilomycin A or comparator were diluted in the appropriate medium to give a maximum starting concentration of 50 µg / ml in the assay. 50 µl of medium was dispensed in each well in columns two to 12 of a 96-well plate. 100 µl of the appropriate test compound solution (100 µg / ml) was dispensed into each well of column one. In the case of M. catarrhalis,

M. haemolytica and P. multocida these values were doubled (a final test volume of 200 µl was used). Serial half dilutions were made from column one to column 10 to give a concentration range of 50 to 0.1 µg / ml of each compound in the assay. Columns 11 and 12 served as controls for positive culture (without drug or test article, inoculum added) and negative (without drug, test article or inoculum added), respectively.

Addition of bacterial strains. 50 µl of each inoculum was added to the appropriate wells, resulting in a final volume of 100 µl consisting of 50 µl of diluted compound or diluents and 50 µl of inoculum or broth alone.

The minimum bactericidal concentrations (MBC) were established by extending the MIC procedure to the evaluation of bactericidal activity. After 24 hours, 10 µl was removed from the wells, serially diluted and then placed on suitable agar plates. The plates were incubated at 37 ° C overnight. The MBC was read 18 h later as the lowest antibiotic concentration that resulted in a 0.1% survival in the subculture. All experiments were performed in triplicate.

The MBCs were also determined in the presence of 50% and 95% (v / v) human serum to assess protein binding.

Bactericidal effects of odilomycin on the growth of S. aureus and P. aeruginosa

Bacterial destruction curves were carried out by inoculating S. aureus ATCC 13709 and P. aeruginosa ATCC 27853 with a concentration of Odilomycin A equal to four times the MIC. Vancomycin or polymyxin was used at a concentration four times their MIC. The inoculums of S.aureus and P.aeruginosa were prepared from colonies grown overnight in MHB. Antibiotic concentrations in the flask were adjusted in MHB according to the desired concentration. The culture tubes containing 10 ml were inoculated with S. aureus or P. aeruginosa at an inoculum of approximately 105 CFU / ml. The samples were removed and the bacteria were counted at 0, 1, 2, 3, 4, 6 and 24 h of incubation at 37 ° C. Thus, after vortexing the culture tubes, two 50 µl samples were removed and serially diluted with MHB. After each dilution stage, 20 µl were plated on LB agar plates, which were incubated for 24 h at 37 ° C. After that, the colonies were counted and re-extrapolated to the original volume to determine the initial concentration (CFU / ml).

In vitro biological properties

Odilomycin A was analyzed to determine its antimicrobial activity against a wide range of bacteria

5 involved in nosocomial and animal infection. It has a broad spectrum of antibacterial activity against gram-positive and gram-negative pathogens (table 8). With respect to gram-positive bacteria, the antibacterial activities of odilomycin A are potent, with MICs lower than 1 µg / ml against S. aureus strains,

S. epidermidis and B. subtilis, including against some multi-resistant clinical isolates (table 9). A weak antibacterial activity or lack thereof was observed against E. faecalis, E. faecium, S. pneumoniae and S.

10 pyogenes With regard to gram-negative bacteria, the antibacterial activities of odilomycin A are potent against K. pneumoniae and K. oxytoca, and are good, with MICs below 10 µg / ml, against strains of A. baumannii, E cloacae, E. coli, M. catarrhalis, P. aerwginosa and S. maltophilia, including some multi-resistant clinical isolates (table 8, table 9).

15 Table 8. Antibacterial activity of Odilomycins A, B, C

Bacteria

Strain MIC (µg / ml)

Odilomycin A

Odilomycin B Odilomycin C Control

Gram (-)

A. baumannii

ATCC BAA747 6.25 - - <0,1a

B. fragilis

ATCC 25238 1.56 - - 0.39b

E. cloacae *

11370 1.56 - - 0.78c

E. coli

ATCC 25922 3.13 - - 0.39c

E. coli CTX-M14 *

BCE7 3.13 - - 0.39c

E. coli CTX-M15 *

MEC23 3.13 - - 0.39c

E. coli TEM *

MEC12 1.56 - - 0.39c

K. pneumoniae

ATCC 43816 0.78 - - 0.39c

K. pneumoniae KPC *

2475 1.56 - - 0.78c

K. oxytoca *

NEB9 0.39 - - 0.39c

M. haemolytica

ATCC 33396 50.0 - - <0,1a

M. catarrhalis

ATCC 25238 1.56 - - <0,1a

P. multocida

ATCC 12945 25.0 - - <0,1a

P. aeruginosa

ATCC 27853 3.13 3.13 6.25 0.39c

S. maltophilia

ATCC 13637 6.25 - - <0,1a

H. influenzae

ATCC 49766 12.5 - - <0,1a

Gram (+)

B. subtilis

DSM 347 <0.20 - - 0.20d

E. faecalis

ATCC 29212 > 50.0 - - 0.78a

E. faecium

ATCC 700221 25.0 - - > 50.0a

S. pneumoniae

ATCC 49619 > 50.0 - - 0.39a

S. pyogenes

ATCC 12384 50.0 - - 0.20a

S. aureus

ATCC 13709 0.39 0.39 0.78 0.78d

S. aureus

ATCC 25923 0.39 0.39 0.78 0.78d

S. aureus USA300

ATCC BAA1556 0.78 0.78 1.56 0.78d

S. epidermidis

ATCC 12228 <0.20 - - 0.20d

P. acnes

ATCC 6919 3.13 - - 0.39a

* clinical isolates from the University Hospital of Nimes; aciprofloxacin; bmetronidazole; cpolimixin; dvancomycin

Table 9. Odilomycin A antibacterial activity against multidrug-resistant clinical isolates.

P. aeruginosa

S. aureus

Antibacterial 1

561 8 40168 1 3517 0 4216 2 51223 2 2330 5 1666 6 2068 1 2184 0 2036 4

Pip./Taz.a

S S S S R - - - - -

Ticar./CAb

S R R R R - - - - -

Erythromycin n

R R R R R R R S S R

Cefepim

S S S S S - - - - -

Cefoxitin

- - - - - I R R S R

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